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Frederickx, Wout; Rocha, Susana; Fujita, Yasuhiko; Kennes, Koen; Keersmaecker, Herlinde de; Feyter, Steven de; Uji-i, Hiroshi; Vanderlinden, Willem (2018): Orthogonal Probing of Single-Molecule Heterogeneity by Correlative Fluorescence and Force Microscopy. In: ACS Nano, Vol. 12, No. 1: pp. 168-177
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Correlative imaging by fluorescence and force microscopy is an emerging technology to acquire orthogonal information at the nanoscale. Whereas atomic force microscopy excels at resolving the envelope structure of nanoscale specimens, fluorescence microscopy can detect specific molecular labels, which enables the unambiguous recognition of molecules in a complex assembly. Whereas correlative imaging at the micrometer scale has been established, it remains challenging to push the technology to the single-molecule level. Here, we used an integrated setup to systematically evaluate the factors that influence the quality of correlative fluorescence and force microscopy. Optimized data processing to ensure accurate drift correction and high localization precision results in image registration accuracies of similar to 25 nm on organic fluorophores, which represents a 2-fold improvement over the state of the art in correlative fluorescence and force microscopy. Furthermore, we could extend the Atto532 fluorophore bleaching time similar to 2-fold, by chemical modification of the supporting mica surface. In turn, this enables probing the composition of macromolecular complexes by stepwise photobleaching with high confidence. We demonstrate the performance of our method by resolving the stoichiometry of molecular subpopulations in a heterogeneous EcoRV-NA nucleoprotein ensemble.